Sphingosine-1-phosphate lyase polypeptides, polynucleotides and modulating agents and methods of use therefor

ABSTRACT

Compositions, methods and kits for diagnosing and treating cancer are provided. Therapeutic compositions may comprise agents that modulate the expression or activity of a sphingosine-1-phosphate lyase (SPL). Such compositions may be administered to a mammal afflicted with cancer. Diagnostic methods and kits may employ an agent suitable for detecting alterations in endogenous SPL. Such methods and kits may be used to detect the presence of a cancer or to evaluate the prognosis of a known disease. SPL polypeptides, polynucleotides and antibodies are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.08/939,309 filed Sep. 29, 1997, now U.S. Pat. No. 6,423,527, whichapplication is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The present invention relates generally to cancer detection and therapy.The invention is more particularly related to sphingosine-1-phosphatelyase polynucleotides and polypeptides, and to agents that modulate theexpression and/or activity of such polypeptides. Such agents may beused, for example, to diagnose and/or treat cancers such as breastcancer.

BACKGROUND OF THE INVENTION

Breast cancer is a significant health problem for women in the UnitedStates and throughout the world. Although advances have been made indetection and treatment of the disease, breast cancer remains the mostcommon form of cancer, and the second leading cause of cancer death, inAmerican women. Among African-American women and women between 15 and 54years of age, breast cancer is the leading cause of cancer death. Oneout of every eight women in the United States will develop breastcancer, a risk which has increased 52% during 1950–1990. In 1994, it isestimated that 182,000 new cases of female breast cancer were diagnosed,and 46,000 women died from the disease.

No vaccine or other universally successful method for the prevention ortreatment of breast cancer is currently available. Management of thedisease currently relies on a combination of early diagnosis (throughroutine breast screening procedures) and aggressive treatment, which mayinclude one or more of a variety of treatments such as surgery,radiotherapy, chemotherapy and hormone therapy. The course of treatmentfor a particular breast cancer is often selected based on a variety ofprognostic parameters, including an analysis of specific tumor markers.However, the use of established markers often leads to a result that isdifficult to interpret.

With current therapies, tumor invasiveness and metastasis is a criticaldeterminant in the outcome for breast cancer patients. Although the fiveyear survival for women diagnosed with localized breast cancer is about90%, the five year survival drops to 18% for women whose disease hasmetastasized. Present therapies are inadequate for inhibiting tumorinvasiveness for the large population of women with this severe disease.

Accordingly, improvements are needed in the treatment, diagnosis andprevention of breast cancer. The present invention fulfills this needand further provides other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compositions and methodsfor the diagnosis and therapy of cancer. Within one aspect, the presentinvention provides isolated polynucleotides comprising a sequenceselected from the group consisting of. (a) a sequence recited in SEQ IDNO:1; (b) a sequence recited in SEQ ID NO:3; (c) nucleotide sequencesthat hybridize to a polynucleotide complementary to either of theforegoing sequences under moderately stringent conditions, wherein thenucleotide sequences encode polypeptides having sphingosine-1-phosphatelyase activity; and (d) nucleotide sequences that encode a polypeptideencoded by any of the foregoing sequences.

Within a related aspect, an isolated polynucleotide is provided thatencodes a polypeptide recited in SEQ ID NO:2, or a variant of such apolypeptide that has sphingosine-1-phosphate lyase activity. In anotherrelated aspect, an isolated polynucleotide comprising a sequence recitedin SEQ ID NO:4, or a variant of such a polypeptide that hassphingosine-1-phosphate lyase activity, is provided.

Recombinant expression vectors comprising any of the foregoingpolynucleotides, and host cells transformed or transfected with suchexpression vectors, are also provided.

Within further aspects, SPL polypeptides are provided. Such polypeptidesmay be encoded by any of the foregoing polynucleotides. Alternatively, apolypeptide may comprise an amino acid sequence recited in SEQ ID NO:2or 4, or a variant thereof, wherein the polypeptide hassphingosine-1-phosphate lyase activity.

Within a further aspect, the present invention provides isolatedpolynucleotides comprising at least 100 nucleotides complementary to asequence recited in SEQ ID NO:1 or 3.

Within other aspects, methods are provided for preparing asphingosine-1-phosphate lyase, comprising culturing a host celltransformed or transfected with a polynucleotide as described aboveunder conditions promoting expression of the polynucleotide andrecovering a sphingosine-1-phosphate lyase.

In further aspects, the present invention provides methods foridentifying an agent that modulates sphingosine-1-phosphate lyaseactivity. In one such aspect, the method comprises: (a) contacting acandidate agent with cells that express sphingosine-1-phosphate lyase;and (b) subsequently measuring the level of sphingosine-1-phosphatelyase or mRNA encoding sphingosine-1-phosphate lyase in the cells,relative to a predetermined level in the absence of candidate agent.Within another such aspect, the method comprises: (a) contacting acandidate agent with a polypeptide comprising a sequence recited in anyone of SEQ ID NOs: 2, 4, 6 or 8, or a variant of such a sequence havingsphingosine-1-phosphate lyase activity, wherein the step of contactingis carried out under conditions and for a time sufficient to allow thecandidate modulator to interact with the polypeptide; and (b)subsequently measuring the ability of the polypeptide to degradesphingosine-1-phosphate or a derivative thereof, relative to an abilityin the absence of candidate agent. The step of contacting may beperformed by incubating a cell expressing the polypeptide with thecandidate modulator, and the step of measuring the ability to degradesphingosine-1-phosphate may be performed using an in vitro assay and acellular extract.

The present invention further provides pharmaceutical compositionscomprising an agent that modulates sphingosine-1-phosphate lyaseactivity in combination with a pharmaceutically acceptable carrier. Suchagents preferably inhibit sphingosine-1-phosphate lyase activity. Suchinhibition may be achieved by inhibiting expression of an endogenous SPLgene, or by inhibiting the ability of an endogenous SPL to degradesphingosine-1-phosphate. Within certain preferred embodiments, amodulating agent comprises a polynucleotide or an antibody or anantigen-binding fragment thereof.

Within still further aspects, the present invention provides methods formodulating sphingosine-1-phosphate activity, comprising contacting asphingosine-1-phosphate lyase with an effective amount of an agent thatmodulates sphingosine-1-phosphate lyase activity, wherein the step ofcontacting is performed under conditions and for a time sufficient toallow the agent and the sphingosine-1-phosphate lyase to interact. Tomodulate sphingosine-1-phosphate lyase activity in a cell, a cellexpressing sphingosine-1-phosphate may be contacted with such an agent.

Within related aspects, the present invention provides methods forinhibiting the growth of a cancer cell, comprising contacting a cancercell with an agent that inhibits sphingosine-1-phosphate lyase activity.In a preferred embodiment, the cancer cell is a breast cancer cell.

The present invention also provides methods for inhibiting thedevelopment and/or metastasis of a cancer in a mammal, comprisingadministering to a mammal an agent that inhibits sphingosine-1-phosphatelyase activity. Within certain embodiments, an agent may comprise, or belinked to, a targeting component, such as an anti-tumor antibody or acomponent that binds to an estrogen receptor.

Within other aspects, methods for diagnosing cancer in a mammal areprovided, comprising detecting an alteration in an endogenoussphingosine-1-phosphate lyase gene in a sample obtained from a mammal,and therefrom diagnosing a cancer in the mammal. In certain embodimentsthe cancer is breast cancer and the sample is a breast tumor biopsy.

In related aspects, the present invention provides methods forevaluating a cancer prognosis, comprising determining the presence orabsence of an alteration in an endogenous sphingosine-1-phosphate lyasegene in a sample obtained from a mammal afflicted with cancer, andtherefrom determining a prognosis.

The present invention further provides isolated antibodies that bind toa polypeptide having a sequence recited in any one of SEQ ID NOs: 2, 4or 6. Such antibodies may be polyclonal or monoclonal, and may inhibitthe ability of a polypeptide having a sequence recited in any one of SEQID NOs: 2, 4 or 6 to degrade sphingosine-1-phosphate.

In still further aspects, the present invention provides methods fordetecting sphingosine-1-phosphate lyase in a sample, comprising: (a)contacting a sample with an antibody as described above under conditionsand for a time sufficient to allow the antibody to bind tosphingosine-1-phosphate lyase; and (b) detecting in the sample thepresence of sphingosine-1-phosphate lyase bound to the antibody.

Kits for use in the above methods are also provided. A kit for detectingsphingosine-1-phosphate lyase in a sample comprises an antibody asdescribed above and a buffer or detection reagent. A kit for detectingan alteration in a sphingosine-1-phosphate gene in a sample comprises apolynucleotide and a detection reagent.

Within further aspects, the present invention provides transgenicanimals in which sphingosine-1-phosphate lyase activity is reduced, andcell lines derived from such transgenic animals.

These and other aspects of the present invention will become apparentupon reference to the following detailed description and attacheddrawings. All references disclosed herein are hereby incorporated byreference in their entirety as if each was incorporated individually.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A–1D present the sequence of a S. cerevisiae polynucleotide (SEQID NO: 7) encoding a representative SPL polypeptide (SEQ ID NO: 8).

FIGS. 2A–2C present the sequence of a C. elegans polynucleotide (SEQ IDNO: 5) encoding a representative SPL polypeptide (SEQ ID NO: 6).

FIGS. 3A–3C present the sequence of a Mus musculus polynucleotide (SEQID NO: 1) encoding a representative SPL polypeptide (SEQ ID NO: 2).

FIGS. 4A and 4B presents a comparison of the endogenous SPL genomicsequences from C. elegans (SEQ ID NO: 6), yeast (SEQ ID NO: 8) and mouse(SEQ ID NO: 2).

FIG. 5 is a photograph showing the growth of yeast cells grown tosaturation in liquid culture and then plated on YPD with (top plate) andwithout (lower plate) 50 μM sphingosine. On each plate, the top row ofcells is BST1Δ (JS16, which is a variation of SGP3 (leu2-3,112 trp1ura3-52 his3 ade8 rasI::HIS3) in which the BST1 gene has been replacedby a G418-resistant marker, NEO). The second row is JS16 transformedwith vector alone. The third row and the bottom two rows (mBST1) showJS60 cells (JS16[pYES-mouseSPL]) and the fourth row (ceBST1) shows JS61cells (JS16[pYES2-C. elegansBST1]). The fifth row on each plate(BST1-WT) shows the growth of the wildtype SGP3 strain.

FIG. 6A is an autoradiogram showing the products of an SPL assayperformed on extracts obtained from JS16 transformed withJS29=pYES2-yeast BST1 (ytBST1), JS60=pYES2-mouseSPL (mBST1) or pYES2without insert (vehicle control). FIG. 6B is a histogram depicting theactivity in the strains shown in FIG. 6A, as determined by scraping aTLC plate as shown in FIG. 6A and assessing the level of radioactivity.

FIG. 7 is an autoradiogram depicting the results of a Northern blotanalysis of the level of mouse SPL in various mouse tissues, asindicated.

FIGS. 8A–8D present a sequence of a human polynucleotide (SEQ ID NO: 3)encoding a representative SPL polypeptide (SEQ ID NO: 4).

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is generally directed tocompositions and methods for the diagnosis and therapy of cancers suchas breast cancer. The invention is more particularly related tosphingosine-1-phosphate lyase (SPL) polypeptides, which have the abilityto cleave sphingosine-1-phosphate into inactive metabolites, and topolynucleotides encoding such polypeptides. Sphingosine-1-phosphate isan endogenous tumor-suppressor lipid that potently inhibits breastcancer cell growth and invasiveness, while not affecting the growth ofnon-tumor cells (see Sadahira et al., Proc. Natl. Acad. Sci. USA89:9686–90, 1992). In vivo, SPL catalyzes the cleavage ofsphingosine-1-phosphate at the C₂₋₃ carbon bond to yield a long chainaldehyde and ethanolamine phosphate, the final step in the degradationof all higher order sphingolipids. Agents that decrease the expressionor activity of endogenous SPL polypeptides are encompassed by thepresent invention. Such modulating agents may be identified usingmethods described herein and used, for example, in cancer therapy. Ithas also been found, within the context of the present invention, thatthe detection of alterations in an endogenous SPL sequence can be usedto diagnose cancer, and to assess the prognosis for recovery. Thepresent invention further provides such diagnostic methods and kits.

As used herein, the term “polypeptide” encompasses amino acid chains ofany length, including full length endogenous (i.e., native) SPL proteinsand variants of endogenous sequences. “Variants” are polypeptides thatdiffer in sequence from a native SPL only in substitutions, deletionsand/or other modifications, such that the variant retains SPL activity,which may be determined using a representative method described herein.Within an SPL polypeptide variant, amino acid substitutions arepreferably made at no more than 50% of the amino acid residues in thenative polypeptide, and more preferably at no more than 25% of the aminoacid residues. Such substitutions are preferably conservative. Aconservative substitution is one in which an amino acid is substitutedfor another amino acid that has similar properties, such that oneskilled in the art of peptide chemistry would expect the secondarystructure and hydropathic nature of the polypeptide to be substantiallyunchanged. In general, the following, amino acids represent conservativechanges: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser,tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5)phe, tyr, trp, his. Substitutions, deletions and/or amino acid additionsmay be made at any location(s) in the polypeptide, provided that themodification does not diminish the SPL activity of the variant. Thus, avariant may comprise only a portion of a native SPL sequence. Inaddition, or alternatively, variants may contain additional amino acidsequences (such as, for example, linkers, tags and/or ligands),preferably at the amino and/or carboxy termini. Such sequences may beused, for example, to facilitate purification, detection or cellularuptake of the polypeptide.

The SPL activity of an SPL polypeptide may generally be assessed usingan in vitro assay that detects the degradation of labeled substrate(i.e., sphingosine-1-phosphate, or a derivative thereof). Within suchassays, pyridoxal 5′-phosphate is a requirement for SPL activity. Inaddition, the reaction generally proceeds optimally at pH 7.4–7.6 andrequires chelators due to sensitivity toward heavy metal ions. Thesubstrate should be a D-erythro isomer, but in derivatives ofsphingosine-1-phosphate the type and chain length of sphingoid base mayvary. In general, an assay as described by Van Veldhoven and Mannaerts,J. Biol. Chem. 266:12502–07, 1991 may be employed. Briefly, a solution(e.g., a cellular extract) containing the polypeptide may be incubatedwith 40 μM substrate at 37° C. for 1 hour in the presence of, forexample, 50 mM sucrose, 100 mM K-phosphate buffer pH 7.4, 25 mM NaF,0.1% (w/v) Triton X-100, 0.5 mM EDTA, 2 mM DTT, 0.25 mM pyridoxalphosphate. Reactions may then be terminated and analyzed by thin-layerchromatography to detect the formation of labeled fatty aldehydes andfurther metabolites. In general, a polypeptide has SPL activity if,within such an assay: (1) the presence of 2–50 μg polypeptide (or 0.1–10mg/mL) results in a statistically significant increase in the level ofsubstrate degradation, preferably a two-fold increase, relative to thelevel observed in the absence of polypeptide; and (2) the increase inthe level of substrate degradation is pyridoxal 5′-phosphate dependent.

Within certain embodiments, an in vitro assay for SPL activity may beperformed using cellular extracts prepared from cells that express thepolypeptide of interest. Preferably, in the absence of a gene encodingan SPL polypeptide, such cells do not produce a significant amount ofendogenous SPL (i.e., a cellular extract should not contain a detectableincrease in the level of SPL, as compared to buffer alone withoutextract). It has been found, within the context of the presentinvention, that yeast cells containing deletion of the SPL gene (BST1)are suitable for use in evaluating the SPL activity of a polypeptide.bst1Δ cells can be generated from S. cerevisiae using standardtechniques, such as PCR, as described herein. A polypeptide to be testedfor SPL activity may then be expressed in bst1Δ cells, and the level ofSPL activity in an extract containing the polypeptide may be compared tothat of an extract prepared from cells that do not express thepolypeptide. For such a test, a polypeptide is preferably expressed on ahigh-copy yeast vector (such as pYES2, which is available fromInvitrogen) yielding more than 20 copies of the gene per cell. Ingeneral, a polypeptide has SPL activity if, when expressed using such avector in a bst1Δ cell, a cellular extract results in a two-foldincrease in substrate degradation over the level observed for an extractprepared from cells not expressing the polypeptide.

A further test for SPL activity may be based upon functionalcomplementation in the bst1Δ strain. It has been found, within thecontext of the present invention, that bst1Δ cells are highly sensitiveto D-erythro-sphingosine. In particular, concentrations as low as 10 μMsphingosine completely inhibit the growth of bst1Δ cells. Such a levelof sphingosine has no effect on the growth of wildtype cells. Apolypeptide having SPL activity as provided above significantlydiminishes (i.e., by at least two fold) the sphingosine sensitivity whenexpressed on a high-copy yeast vector yielding more than 20 copies ofthe gene per cell.

In general, SPL polypeptides, and polynucleotides encoding suchpolypeptides, may be prepared using any of a variety of techniques thatare well known in the art. For example, a DNA sequence encoding nativeSPL may be prepared by amplification from a suitable cDNA or genomiclibrary using, for example, polymerase chain reaction (PCR) orhybridization techniques. Libraries may generally be prepared andscreened using methods well known to those of ordinary skill in the art,such as those described in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor,N.Y., 1989. cDNA libraries may be prepared from any of a variety ofsources known to contain enzymes having SPL activity. SPL activity isubiquitous with regard to species and mammalian tissues, with theexception of platelets, in which SPL activity is notably absent. In rattissues, the highest levels of activity have been demonstrated inintestinal mucosa, liver and Harderian gland, with low activity inskeletal muscle and heart. Activity has also been demonstrated in anumber of human (hepatoma cell line HB 8065, cervical carcinoma HeLa),mouse (hepatoma line BW1, mouse embryo 3T3-L1, Swiss 3T3 cells) andother cell lines, as well as in human cultured fibroblasts. PreferredcDNA libraries may prepared from human liver, intestine or brain tissuesor cells. Other libraries that may be employed will be apparent to thoseof ordinary skill in the art. Primers for use in amplification may bereadily designed based on the sequence of a native SPL polypeptide orpolynucleotide, as provided herein.

Alternatively, an endogenous SPL gene may be identified using a screenfor cDNAs that complement the BST1 deletion in yeast. A cDNA expressionlibrary may be generated using a regulatable yeast expression vector(e.g, pYES, which is available from Invitrogen, Inc.) and standardtechniques. A yeast bst1Δ strain may then be transformed with the cDNAlibrary, and endogenous cDNAs having the ability to functionallycomplement the yeast lyase defect (i.e., restore the ability to grow inthe presence of D-erythro-sphingosine) may be isolated.

An endogenous SPL gene may also be identified based on cross-reactivityof the protein product with anti-SPL antibodies, which may be preparedas described herein. Such screens may generally be performed usingstandard techniques (see Huynh et al., “Construction and Screening cDNALibraries in λgt11,” in D. M. Glover, ed., DNA Cloning: A PracticalApproach, 1:49–78, 1984 (IRL Press, Oxford)).

Polynucleotides encompassed by the present invention include DNA and RNAmolecules that comprise an endogenous SPL gene sequence. Suchpolynucleotides include those that comprise a sequence recited in anyone of SEQ ID NOs:1, 3, 5 and 7. Also encompassed are otherpolynucleotides that encode an SPL amino acid sequence provided in anyone of SEQ ID NOs: 2, 4, 6 and 8, as well as polynucleotides that encodevariants of a native SPL sequence that retain SPL activity.Polynucleotides that are substantially homologous to a sequencecomplementary to an endogenous SPL gene are also within the scope of thepresent invention. “Substantial homology,” as used herein refers topolynucleotides that are capable of hybridizing under moderatelystringent conditions to a polynucleotide complementary to a sequenceprovided in SEQ ID NO:1 or SEQ ID NO:3, provided that the encoded SPLpolypeptide variant retains SPL activity. Suitable moderately stringentconditions include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0 mMEDTA (pH 8.0); hybridizing at 50–65° C., 5×SSC, overnight; followed bywashing twice at 65° C. for 20 minutes with each of 2×, 0.5× and 0.2×SSCcontaining 0.1% SDS. Nucleotide sequences that, because of codedegeneracy, encode a polypeptide encoded by any of the above sequencesare also encompassed by the present invention.

Polypeptides of the present invention may be prepared by expression ofrecombinant DNA encoding the polypeptide in cultured host cells.Preferably, the host cells are bacteria, yeast, insect or mammaliancells, and more preferably the host cells are S. cerevisiae bst1Δ cells.The recombinant DNA may be cloned into any expression vector suitablefor use within the host cell and transfected into the host cell usingtechniques well known to those of ordinary skill in the art. A suitableexpression vector contains a promoter sequence that is active in thehost cell. A tissue-specific or conditionally active promoter may alsobe used. Preferred promoters express the polypeptide at high levels.

Optionally, the construct may contain an enhancer, a transcriptionterminator, a poly(A) signal sequence, a bacterial or mammalian originof replication and/or a selectable marker, all of which are well knownin the art. Enhancer sequences may be included as part of the promoterregion or separately. Transcription terminators are sequences that stopRNA polymerase-mediated transcription. The poly(A) signal may becontained within the termination sequence or incorporated separately. Aselectable marker includes any gene that confers a phenotype on the hostcell that allows transformed cells to be identified. Such markers mayconfer a growth advantage under specified conditions. Suitableselectable markers for bacteria are well known and include resistancegenes for ampicillin, kanamycin and tetracycline. Suitable selectablemarkers for mammalian cells include hygromycin, neomycin, genes thatcomplement a deficiency in the host (e.g., thymidine kinase and TK⁻cells) and others well known in the art. For yeast cells, one suitableselectable marker is URA3, which confers the ability to grow on mediumwithout uracil.

DNA sequences expressed in this manner may encode a native SPLpolypeptide (e.g., human), or may encode portions or other variants ofnative SPL polypeptide. DNA molecules encoding variants of a native SPLmay generally be prepared using standard mutagenesis techniques, such asoligonucleotide-directed site-specific mutagenesis, and sections of theDNA sequence may be removed to permit preparation of truncatedpolypeptides.

To generate cells that express a polynucleotide encoding an SPLpolypeptide, cells may be transfected using any of a variety oftechniques known in the art. Such transfection may result in stabletransformants or may be transient. One suitable transfection techniqueis electroporation, which may be performed on a variety of cell types,including mammalian cells, yeast cells and bacteria, using commerciallyavailable equipment. Optimal conditions for electroporation (includingvoltage, resistance and pulse length) are experimentally determined forthe particular host cell type, and general guidelines for optimizingelectroporation may be obtained from manufacturers. Other suitablemethods for transfection will depend upon the type of cell used (e.g.,the lithium acetate method for yeast), and will be apparent to those ofordinary skill in the art. Following transfection, cells may bemaintained in conditions that promote expression of the polynucleotidewithin the cell. Appropriate conditions depend upon the expressionsystem and cell type, and will be apparent to those skilled in the art.

SPL polypeptides may be expressed in transfected cells by culturing thecell under conditions promoting expression of the transfectedpolynucleotide. Appropriate conditions will depend on the specific hostcell and expression vector employed, and will be readily apparent tothose of ordinary skill in the art. For commercially availableexpression vectors, the polypeptide may generally be expressed accordingto the manufacturer's instructions. For certain purposes, expressedpolypeptides of this invention may be isolated in substantially pureform. Preferably, the polypeptides are isolated to a purity of at least80% by weight, more preferably to a purity of at least 95% by weight,and most preferably to a purity of at least 99% by weight. In general,such purification may be achieved using, for example, the standardtechniques of ammonium sulfate fractionation, SDS-PAGE electrophoresis,and/or affinity chromatography.

The present invention further provides antibodies that bind to an SPLpolypeptide. Antibodies may function as modulating agents (as discussedfurther below) to inhibit or block SPL activity in vivo. Alternatively,or in addition, antibodies may be used within screens for endogenous SPLpolypeptides or modulating agents, for purification of SPL polypeptides,for assaying the level of SPL within a sample and/or for studies of SPLexpression. Such antibodies may be polyclonal or monoclonal, and aregenerally specific for one or more SPL polypeptides and/or one or morevariants thereof. Within certain preferred embodiments, antibodies arepolyclonal.

Antibodies may be prepared by any of a variety of techniques known tothose of ordinary skill in the art (see, e.g, Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).In one such technique, an immunogen comprising an SPL polypeptide orantigenic portion thereof is initially injected into a suitable animal(e.g., mice, rats, rabbits, sheep and goats), preferably according to apredetermined schedule incorporating one or more booster immunizations.The use of rabbits is preferred. To increase immunogenicity, animmunogen may be linked to, for example, glutaraldehyde or keyholelimpet hemocyanin (KLH). Following injection, the animals are bledperiodically to obtain post-immune serum containing polyclonal anti-SPLantibodies. Polyclonal antibodies may then be purified from suchantisera by, for example, affinity chromatography using an SPLpolypeptide or antigenic portion thereof coupled to a suitable solidsupport. Such polyclonal antibodies may be used directly for screeningpurposes and for Western blots.

More specifically, an adult rabbit (e.g., NZW) may be immunized with 10μg purified (e.g., using a nickel-column) SPL polypeptide emulsified incomplete Freund's adjuvant (1:1 v/v) in a volume of 1 mL. Immunizationmay be achieved via injection in at least six different subcutaneoussites. For subsequent immunizations, 5 μg of an SPL polypeptide may beemulsified in in complete Freund's adjuvant and injected in the samemanner. Immunizations may continue until a suitable serum antibody titeris achieved (typically a total of about three immunizations). The rabbitmay be bled immediately before immunization to obtain pre-immune serum,and then 7–10 days following each immunization.

For certain embodiments, monoclonal antibodies may be desired.Monoclonal antibodies may be prepared, for example, using the techniqueof Kohler and Milstein, Eur. J. Immunol. 6:511–519, 1976, andimprovements thereto. Briefly, these methods involve the preparation ofimmortal cell lines capable of producing antibodies having the desiredspecificity (i.e., reactivity with the polypeptide of interest). Suchcell lines may be produced, for example, from spleen cells obtained froman animal immunized as described above. The spleen cells are thenimmortalized by, for example, fusion with a myeloma cell fusion partner,preferably one that is syngeneic with the immunized animal. For example,the spleen cells and myeloma cells may be combined with a nonionicdetergent for a few minutes and then plated at low density on aselective medium that supports the growth of hybrid cells, but notmyeloma cells. A preferred selection technique uses HAT (hypoxanthine,aminopterin, thymidine) selection. After a sufficient time, usuallyabout 1 to 2 weeks, colonies of hybrids are observed. Single coloniesare selected and tested for binding activity against the polypeptide.Hybridomas having high reactivity and specificity are preferred.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies. In addition, various techniques may be employed toenhance the yield, such as injection of the hybridoma cell line into theperitoneal cavity of a suitable vertebrate host, such as a mouse.Monoclonal antibodies may then be harvested from the ascites fluid orthe blood. Contaminants may be removed from the antibodies byconventional techniques, such as chromatography, gel filtration,precipitation, and extraction.

As noted above, the present invention provides agents that modulate,preferably inhibit, the expression (transcription or translation),stability and/or activity of an SPL polypeptide. To identify such amodulating agent, any of a variety of screens may be performed.Candidate modulating agents may be obtained using well known techniquesfrom a variety of sources, such as plants, fungi or libraries ofchemicals, small molecules or random peptides. Antibodies that bind toan SPL polypeptide, and anti-sense polynucleotides that hybridize to apolynucleotides that encodes an SPL, may be candidate modulating agents.Preferably, a modulating agent has a minimum of side effects and isnon-toxic. For some applications, agents that can penetrate cells arepreferred.

Screens for modulating agents that decrease SPL expression or stabilitymay be readily performed using well known techniques that detect thelevel of SPL protein or mRNA. Suitable assays include RNAse protectionassays, in situ hybridization, ELISAs, Northern blots and Western blots.Such assays may generally be performed using standard methods (seeSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratories, Cold Spring Harbor, N.Y., 1989). For example, todetect mRNA encoding SPL, a nucleic acid probe complementary to all or aportion of the SPL gene sequence may be employed in a Northern blotanalysis of mRNA prepared from suitable cells. To detect SPL protein, areagent that binds to the protein (typically an antibody, as describedherein) may be employed within an ELISA or Western assay. Followingbinding, a reporter group suitable for direct or indirect detection ofthe reagent is employed (i.e., the reporter group may be covalentlybound to the reagent or may be bound to a second molecule, such asProtein A, Protein G, immunoglobulin or lectin, which is itself capableof binding to the reagent). Suitable reporter groups include, but arenot limited to, enzymes (e.g., horseradish peroxidase), substrates,cofactors, inhibitors, dyes, radionuclides, luminescent groups,fluorescent groups and biotin. Such reporter groups may be used todirectly or indirectly detect binding of the reagent to a samplecomponent using standard methods known to those of ordinary skill in theart.

To use such assays for identifying a modulating agent, the level of SPLprotein or MRNA may be evaluated in cells treated with one or morecandidate modulating agents. An increase or decrease in SPL levels maybe measured by evaluating the level of SPL MRNA and/or protein in thepresence and absence of candidate modulating agent. For example, anantisense modulating agent may be evaluated by assaying the effect onSPL levels. Suitable cells for use in such assays include the breastcancer cell lines MCF-7 (ATCC Accession Number HTB-22) and MDA-MB-231(ATCC Accession Number HTB-26). A candidate modulator may be tested bytransfecting the cells with a polynucleotide encoding the candidate andevaluating the effect of expression of the-polynucleotide on SPL levels.Alternatively, the cells may be contacted with a candidate modulator,typically in an amount ranging from about 10 μM to about 10 mM. Acandidate that results in a statistically significant change in thelevel of SPL mRNA and/or protein is a modulating agent.

Alternatively, or in addition, a candidate modulating agent may betested for the ability to inhibit SPL activity, using an in vitro assayas described herein (see Van Veldhoven and Mannaerts, J. Biol. Chem.266:12502–07, 1991) that detects the degradation of labeled substrate(i.e., sphingosine-1-phosphate, or a derivative thereof). Briefly, asolution (e.g., a cellular extract) containing an SPL polypeptide (e.g.,10 nM to about 10 mM) may be incubated with a candidate modulating agent(typically 1 nM to 10 mM, preferably 10 nM to 1 mM) and a substrate(e.g., 40 μM) at 37° C. for 1 hour in the presence of, for example, 50mM sucrose, 100 mM K-phosphate buffer pH 7.4, 25 mM NaF, 0.1% (w/v)Triton X-100, 0.5 mM EDTA, 2 mM DTT, 0.25 mM pyridoxal phosphate.Reactions may then be terminated and analyzed by thin-layerchromatography to detect the formation of labeled fatty aldehydes andfurther metabolites. A modulating agent i(e.g., an antibody) thatinhibits SPL activity results in a statistically significant decrease inthe degradation of sphingosine-1-phosphate, relative to the level ofdegradation in the absence of modulating agent. Such modulating agentsmay be used to inhibit SPL activity in a cell culture or a mammal, asdescribed below.

A modulating agent may additionally comprise, or may be associated with,a targeting component that serves to direct the agent to a desiredtissue or cell type. As used herein, a “targeting component” may be anysubstance (such as a compound or cell) that, when linked to a compoundenhances the transport of the compound to a target tissue, therebyincreasing the local concentration of the compound. Targeting componentsinclude antibodies or fragments thereof, receptors, ligands and othermolecules that bind to cells of, or in the vicinity of, the targettissue. Known targeting components include hormones, antibodies againstcell surface antigens, lectins, adhesion molecules, tumor cell surfacebinding ligands, steroids, cholesterol, lymphokines, fibrinolyticenzymes and other drugs and proteins that bind to a desired target site.In particular, anti-tumor antibodies and compounds that bind to anestrogen receptor may serve as targeting components. An antibodyemployed in the present invention may be an intact (whole) molecule, afragment thereof, or a functional equivalent thereof. Examples ofantibody fragments are F(ab′)2, −Fab′, Fab and F[v] fragments, which maybe produced by conventional methods or by genetic or proteinengineering. Linkage may be via any suitable covalent bond usingstandard techniques that are well known in the art. Such linkage isgenerally covalent and may be achieved by, for example, directcondensation or other reactions, or by way of bi- or multi-functionallinkers.

For in vivo use, a modulating agent as described herein is generallyincorporated into a pharmaceutical composition prior to administration.A pharmaceutical composition comprises one or more modulating agents incombination with a physiologically acceptable carrier. To prepare apharmaceutical composition, an effective amount of one or moremodulating agents is mixed with any pharmaceutical carrier(s) known tothose skilled in the art to be suitable for the particular mode ofadministration. A pharmaceutical carrier may be liquid, semi-liquid orsolid. Solutions or suspensions used for parenteral, intradermal,subcutaneous or topical application may include, for example, a sterilediluent (such as water), saline solution, fixed oil, polyethyleneglycol, glycerine, propylene glycol or other synthetic solvent;antimicrobial agents (such as benzyl alcohol and methyl parabens);antioxidants (such as ascorbic acid and sodium bisulfite) and chelatingagents (such as ethylenediaminetetraacetic acid (EDTA)); buffers (suchas acetates, citrates and phosphates). If administered intravenously,suitable carriers include physiological saline or phosphate bufferedsaline (PBS), and solutions containing thickening and solubilizingagents, such as glucose, polyethylene glycol, polypropylene glycol andmixtures thereof. In addition, other pharmaceutically active ingredients(including other anti-cancer agents) and/or suitable excipients such assalts, buffers and stabilizers may, but need not, be present within thecomposition.

A modulating agent may be prepared with carriers that protect it againstrapid elimination from the body, such as time release formulations orcoatings. Such carriers include controlled release formulations, suchas, but not limited to, implants and microencapsulated delivery systems,and biodegradable, biocompatible polymers, such as ethylene vinylacetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylacticacid and others known to those of ordinary skill in the art.

Administration may be achieved by a variety of different routes,including oral, parenteral, nasal, intravenous, intradermal,subcutaneous or topical. Preferred modes of administration depend uponthe nature of the condition to be treated or prevented. An amount that,following administration, inhibits, prevents or delays the progressionand/or metastasis of a cancer is considered effective. Preferably, theamount administered is sufficient to result in regression, as indicatedby 50% mass or by scan dimensions. The precise dosage and duration oftreatment is a function of the disease being treated and may bedetermined empirically using known testing protocols or by testing thecompositions in model systems known in the art and extrapolatingtherefrom. Controlled clinical trials may also be performed. Dosages mayalso vary with the severity of the condition to be alleviated. Apharmaceutical composition is generally formulated and administered toexert a therapeutically useful effect while minimizing undesirable sideeffects. The composition may be administered one time, or may be dividedinto a number of smaller doses to be administered at intervals of time.For any particular subject, specific dosage regimens may be adjustedover time according to the individual need.

As an alternative to direct administration of a modulating agent, apolynucleotide encoding a modulating agent may be administered. Such apolynucleotide may be present in a pharmaceutical composition within anyof a variety of delivery systems known to those of ordinary skill in theart, including nucleic acid, bacterial and viral expression systems, andcolloidal dispersion systems such as liposomes. Appropriate nucleic acidexpression systems contain the necessary DNA sequences for expression inthe patient (such as a suitable promoter and terminating signal, asdescribed above). The DNA may also be “naked,” as described, forexample, in Ulmer et al., Science 259:1745–49, 1993.

Various viral vectors that can be used to introduce a nucleic acidsequence into the targeted patient's cells include, but are not limitedto, vaccinia or other pox virus, herpes virus, retrovirus, oradenovirus. Techniques for incorporating DNA into such vectors are wellknown to those of ordinary skill in the art. Another delivery system forpolynucleotides is a colloidal dispersion system. Colloidal dispersionsystems include macromolecule complexes, nanocapsules, microspheres,beads, and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, and liposomes. The preparation and use ofliposomes is well known to those of ordinary skill in the art.

Within certain aspects of the present invention, one or more modulatingagents may be used to modulate SPL expression and/or activity in vitro,in a cell or in a mammal. In vitro, an SPL polypeptide may be contactedwith a modulating agent that inhibits SPL activity (e.g., certainantibodies). For use within a cell or a mammal, such modulation may beachieved by contacting a target cell with an effective amount of amodulating agent, as described herein. Administration to a mammal maygenerally be achieved as described above.

As noted above, inhibition of SPL expression and/or activity provides amethod for inhibiting the growth (i.e., proliferation) of a cancer cell,either in culture or in a mammal afflicted with cancer. In vivo, suchinhibition may also be used to inhibit cancer development, progressionand/or metastasis. Accordingly, one or more modulating agents asprovided herein may be administered as described above to a mammal inneed of anti-cancer therapy. Patients that may benefit fromadministration of a modulating agent are those afflicted with cancer.Such patients may be identified based on standard criteria that are wellknown in the art. Within preferred embodiments, a patient is afflictedwith breast cancer, as identified based on tissue biopsy and microscopicevaluation, using techniques well known in the art. In particular,patients whose tumor cells contain a tissue-specific deletion and/oralteration within an endogenous SPL gene may benefit from administrationof a modulating agent, as provided herein.

Within other aspects, the present invention provides methods and kitsfor diagnosing cancer and/or identifying individuals with a risk formetastasis that is higher or lower than average. It has been found,within the context of the present invention, that certain human tumorcells contain an altered SPL gene. In particular, certain brain tumorcells contain a deletion of residues 354 to 433 of the human SPLsequence indicated in FIG. 8 and SEQ ID NO:4. Specific alterationspresent in other tumor cells, such as breast tumor cells, may be readilyidentified using standard techniques, such as PCR. Alterations that maybe associated with a particular tumor include amino acid deletions,insertions, substitutions and combinations thereof. Methods in which thepresence or absence of such an alteration is determined may generally beused to detect cancer and to evaluate the prognosis for a patient knownto be afflicted with cancer.

To detect an altered SPL gene, any of a variety of well-known techniquesmay be used including, but not limited to, PCR and hybridizationtechniques. Any sample that may contain cancerous cells may be assayed.In general, suitable samples are tumor biopsies. Within a preferredembodiment, a sample is a breast tumor biopsy.

Kits for diagnosing or evaluating the prognosis of a cancer generallycomprise reagents for use in the particular assay to be employed. Ingeneral, a kit of the present invention comprises one or more containersenclosing elements, such as probes, reagents or buffers, to be used inan assay. For example, a kit may contain one or more polynucleotideprobes comprising at least 100 nucleotides, and preferably at least 200nucleotides, complementary to an SPL mRNA. Such probe(s) may be used todetect an altered SPL gene by hybridization. For example, a kit maycontain one probe that hybridizes to a region of an SPL gene that is notgenerally altered in tumors (a control) and a second probe thathybridizes to a region commonly deleted in breast cancer. A sample thatcontains mRNA that hybridizes to the first probe, and not to the second(using standard techniques) contains an altered SPL gene. Suitablecontrol probes include probes that hybridize to a portion of the SPLgene outside of the commonly deleted region encoding amino acid resides354 to 433; suitable probes for an altered region include probes thathybridize to a portion of the SPL gene that encodes amino acid residues354 to 433. Alternatively, a kit may comprise one or more primers forPCR analyses, which may be readily designed based upon the sequencesprovided herein by those of ordinary skill in the art. Optionally, a kitmay further comprise one or more solutions, compounds or detectionreagents for use within an assay as described above.

In a related aspect of the present invention, kits for detecting SPL areprovided. Such kits may be designed for detecting the level of SPL ornucleic acid encoding SPL within a sample, or may detect the level ofSPL activity as described herein. A kit for detecting the level of SPL,or nucleic acid encoding SPL, typically contains a reagent that binds tothe SPL protein, DNA or RNA. To detect nucleic acid encoding SPL, thereagent may be a nucleic acid probe or a PCR primer. To detect SPLprotein, the reagent is typically an antibody. The kit may also containa reporter group suitable for direct or indirect detection of thereagent as described above.

Within further aspects, the present invention provides transgenicmammals in which SPL activity is reduced, compared to a wild-typeanimal. Such animals may contain an alteration, insertion or deletion inan endogenous SPL gene, or may contain DNA encoding a modulating agentthat inhibits expression or activity of an SPL gene. Transgenic animalsmay be generated using techniques that are known to those of ordinaryskill in the art. For example, a transgenic animal containing aninsertion or deletion in the coding region for the SPL gene may begenerated from embryonic stem cells, using standard techniques. Suchstem cells may be generated by first identifying the full genomicsequence of the gene encoding the SPL, and then creating an insertion ordeletion in the coding region in embryonic stem cells. Alternatively,appropriate genetically altered embryonic stem cells may be identifiedfrom a bank. Using the altered stem cells, hybrid animals may begenerated with one normal SPL gene and one marked, abnormal gene. Thesehybrids may be mated, and homozygous progeny identified.

Transgenic animals may be used for a variety of purposes, which will beapparent to those of ordinary skill in the art. For example, suchanimals may be used to prepare cell lines from different tissues, usingwell known techniques. Such cell lines may be used, for example, toevaluate the effect of the alteration, and to test various candidatemodulators.

Summary of Sequence Listing SEQ ID NO:1 is cDNA sequence encoding mouseendogenous SPL. SEQ ID NO:2 is amino acid sequence of mouse endogenousSPL. SEQ ID NO:3 is cDNA sequence encoding human endogenous SPL. SEQ IDNO:4 is amino acid sequence of human endogenous SPL. SEQ ID NO:5 is cDNAsequence encoding C. elegans endogenous SPL. SEQ ID NO:6 is amino acidsequence of C. elegans endogenous SPL. SEQ ID NO:7 is cDNA sequenceencoding yeast endogenous SPL. SEQ ID NO:8 is amino acid sequence ofyeast endogenous SPL. SEQ ID NO:9 is cDNA sequence encoding an alteredhuman SPL. SEQ ID NO:10 is amino acid sequence of an altered human SPL.

The following Examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Isolation and Characterization of SPL cDNA from Yeast

This Example illustrates the preparation of an S. cerevisiae cDNAmolecule encoding an endogenous SPL polypeptide.

Wild-type yeast cells (SGP3 (Garrett and Broach, Genes and Dev.3:1336–1348, 1989); leu2-3,112 trp1 ura3-52 his3 ade8 ras1::HIS3) weretransformed with a yeast genomic library carried on the pRS202 high-copyshuttle vector (Sikorski and Heiter, Genetics 122:19–27, 1989)containing a selectable nutritional marker (URA3). pRS202 is a modifiedversion of the pRS306 vector, into which a 2 micron plasmid piece wasinserted. Inserts from this library are approximately 6–8 kb in length.Wild type yeast were transformed with the high copy library as describedby Ito et al., J. Bact. 153:163–68, 1983, selected for uracilprototrophy (i.e., the ability to grow on medium lacking uracil), andtransformants were pooled and replated at a concentration of 10⁶ cellsper plate onto 1 mM D-erythro-sphingosine plates.

Six transformants which grew large colonies on 1 mMD-erythro-sphingosine plates were grown in selective medium, and controlSGP3 colonies were grown in minimal medium, at 30° C. until saturated.Absorbance at 660 nm was used to correct for small variations in cellconcentration between cultures. Serial dilutions were performed, andcells were template-inoculated onto 1 mM D-erythro-sphingosine platesand incubated at 30° C. for 48 hours.

The most highly represented insert, 13-1, was subcloned and sequenced,and named BST1 (bestower of sphingosine tolerance; GenBank accessionnumber U5 1031; Saccharomyces cerevisiae genome database accessionnumber YDR294C). The BST1 nucleotide sequence encodes a previouslyunknown predicted protein of 65,523 kilodaltons and 589 amino acids inlength. This sequence is 23% identical to gadA and gadB, two nearlyidentical E. coli genes encoding glutamate decarboxylase (GAD), apyridoxal-5′-phosphate-dependent enzyme which catalyzes synthesis of theneurotransmitter γ-amino butyric acid. BST1 has been localized to S.cerevisiae chromosome 4. The sequence of BST1 is provided in FIG. 1 andSEQ ID NO:7.

To explore the function of BST1, a deletion strain was created throughhomologous recombination using a NEO selectable marker (Wach et al.,Yeast 10:1793–1808, 1994). Genomic BST1 was replaced with kanMX (Wach etal., Yeast 10:1793–1808, 1994), which confers resistance to G418.Disruption was confirmed using PCR amplification of genomic DNA fromG418 resistant clones, using primers to genomic sequence just 5′ and 3′to the region replaced by the disruption. Deletion of BST1 and allsubsequent biological studies were performed in both SGP3 and in JK93d(Hietman et al., Proc. Natl. Acad. Sci. USA 88:1948–52, 1991); ura3-52leu2-3,112 his4 trp1 rme1). Heterozygous diploids were sporulated, andspores segregated 2:2 for G418 resistance. Both G418 resistant andsensitive progeny were viable, indicating that BST1 is not an essentialgene.

Analysis of GAD activity in cytosolic extracts from wild type, BST1overexpression and bst1Δ strains indicated that BST1 does not encode theS. cervisiae homologue of GAD. However, deletion of BST1 was associatedwith severe sensitivity to D-erythro-sphingosine. Concentrations as lowas 10 μM sphingosine completely inhibited growth of bst1Δ strains buthad no effect on the viability of wild type cells. In comparison to thecontrol strain, the bst1Δ strain also demonstrated greater sensitivityto 100 μM phytosphingosine, the long chain base endogenous to S.cerevisia. No difference between the growth of wild type and BST1overexpression strains on phytosphingosine, which is only minimallytoxic to wild type cells at this concentration, was observed.

To determine whether differences in sphingosine uptake or metabolismwere responsible for these sensitivity differences, BST1 wild type,overexpression and bst1 Δ strains were exposed to [C3-³H]labeledsphingosine (American Radiolabeled Chemical, Inc., St. Louis, Mo.),washed in sterile water and subjected to Bligh-Dyer extractions (Blighand Dyer, Can. J. Buichem. Physiol. 37:911–17, 1959). There were nomajor differences in sphingosine recovery among the three strains.However, the aqueous phase from the bst1Δ strain contained a ten-foldincrease in radioactivity over that of control and BST1 overexpressionstrains. Thin layer chromatography (TLC) analysis of the lipid fractionsin butanol:acetic acid:water (3:1:1) revealed a sphingosine band whichappeared equivalent in each strain.

Radioactive sphingosine-1-phosphate (S-1-P) was also observed in theextracts from the bst1Δ strain, but not in the wild type or BST1overexpression strains. This compound accumulated rapidly, reaching aplateau by 60 minutes. Three separate TLC conditions were used toconfirm the presence of S-1-P. These conditions, along with theresulting RF values, are shown below:

-   -   butanol:water:acetic acid (3:1:1) 0.47    -   chloroform:methanol:water (60:35:8) 0.22    -   chloroform:methanol:water:acetic acid (30:30:2:5) 0.33

Hyperaccumulation of S-1-P and hypersensitivity to D-erythro-sphingosinesuggest a failure to metabolize S-1-P, indicating that BST1 is a yeastSPL. To confirm this identification, lyase activity in BST1 wild type,overexpression and deletion strains were evaluated as described byVeldhoven and Mannaerts, J. Biol. Chem. 266:12502–07, 1991, usingunlabeled D-erythro-dihydrosphingosine-1-phosphate (Biomol, PlymouthMeeting, Pa.) and D-erythro-dihydrosphingosine [4,5-³H]1-phosphate(American Radiolabeled Chemicals, Inc., St. Louis, Mo.). Specificactivity was 100 mCi/mmol. SPL activity was found to correlate with BST1expression, confirming BST1 to be the yeast homologue ofsphingosine-1-phosphate lyase.

These results indication that BST1 is a yeast SPL, and that SPLcatalyzes a rate-limiting step in sphingolipid catabolism. Regulation ofSPL activity may therefore result in regulation of intracellular S-1-Plevels.

Example 2 Isolation and Characterization of SPL cDNA from C. elegans andMouse

This Example illustrates the identification of endogenous SPL cDNAs fromC. elegans and Mus musculus.

Comparison of the yeast BST1 sequence to sequences within the GenBankdatabase identified a full length gene from C. elegans that wasidentified during the systematic sequencing of the C. elegans genome.This sequence was found to encode SPL, and is shown in FIG. 2 and SEQ IDNOs:5 and 6. This and other DNA homology searches described hereinwereperformed via the National Center for Biotechnology Information websiteusing BLAST search program.

Using both S. cerevisiae and C. elegans SPL sequences to search the ESTdatabase, an expressed sequence tag from early embryonic cells of themouse (day 8 embryo, strain C57BL/6J) was identified. The cDNA clonecontaining this putative mouse SPL was purchased from Genome Systems,Inc (St. Louis, Mo.). Completion of the full length cDNA sequencerevealed an 1709 bp open reading frame (FIG. 3 and SEQ ID NOs:1 and 2).This mouse sequence showed significant homology to BST1 and to otherpyridoxal phosphate-binding enzymes such as glutamate decarboxylase,with greatest conservation surrounding the predicted pyridoxalphosphate-binding lysine (FIG. 4). Since the two genes encoding mouseglutamate decarboxylase have been identified previously, and theidentified sequence was unique and had no known function, it was alikely candidate mouse SPL gene.

To confirm the SPL activity of the mouse gene, a two step process wasundertaken. First, the sequence was cloned into the high-copy yeastexpression vector, pYES2 (Invitrogen, Inc., Carlsbad, Calif.), in whichthe gene of interest is placed under control of the yeast GAL promoterand is, therefore, transcriptionally activated by galactose andrepressed by glucose. pYES2 also contains the URA3 gene (which providestransformants the ability to grow in media without uracil) and anampicillin resistance marker and origin of replication functional in E.coli.

The expression vector containing the full-length mouse SPL gene was thenintroduced into the yeast bst1Δ strain which as noted above, isextremely sensitive to D-erythro-sphingosine, as a result of metabolismof sphingosine to S-1-P. S-1-P cannot be further degraded in the absenceof SPL activity and overaccumulates, causing growth inhibition.Transformation was performed using the lithium acetate method (Ito etal., J. Bact. 153:163–68, 1983). Transformants were grown on mediumcontaining 20 g/L galactose and selected for uracil prototrophy.

Transformants were then evaluated for sphingosine resistance. Strains ofinterest were grown to saturation in liquid culture for 2–3 days. Theywere then resuspended in minimal medium, placed in the first row of a96-well plate and diluted serially from 1:2 to 1:4000 across the plate.The cultures were then template inoculated onto a control plate (YPD)and a plate containing minimal synthetic media supplemented with 50 μMD-erythro-sphingosine (Sigma Chemical Co., St. Louis, Mo.) and 0.0015%NP40 (Sigma Chemical Co.). At this concentration of NP40, no effects oncell viability were observed. Plates were incubated at 30° C. for twodays and assessed visually for differences in growth. Transformantscontaining the mouse SPL gene were resistant to sphingosine present ingalactose-containing plates (FIG. 5). A strain transformed with vectoralone remained sensitive to sphingosine. Therefore, the mouse SPL genewas capable of reversing the sphingosine-sensitive phenotype of a yeastbst1Δ strain.

In order to determine whether the mouse SPL gene was able to restorebiochemical SPL activity to the bst1Δ strain, the untransformed bst1Δstrain, and the bst1Δ strain transformed with pYES2 containing eitherBST1 or the putative mouse SPL gene were grown to exponential phase(A₆₀₀=1.0) in either minimal (JS16) or uracil medium containinggalactose as a carbon source. Whole cell extracts were prepared fromeach strain as described above, adjusted for protein concentration, andevaluated for sphingosine phosphate lyase activity as described above,using ³H-dihydrosphingosine-1-phosphate (American RadiolabeledChemicals, Inc., St. Louis, Mo.). Qualitative analysis of product wasperformed by autoradiography. Quantitative measurement was performed byscraping TLC plates and determining radioactivity present using astandard scintillation counter.

The results of the sphingosine phosphate lyase assays are shown in FIGS.6A and 6B. Expression of both the yeast and mouse sequences restored SPLactivity to the bst1Δ strain, whereas vector alone had no effect,confirming the identity of the mouse sequence as SPL.

To determine whether the expression of the mouse SPL transcriptcoincided with previously reported tissue-specific SPL activity in themouse, total RNA was obtained from a variety of mouse tissues and probedwith the complete mouse SPL cDNA sequence. Northern analysis wasperformed as described by Thomas, Proc. Natl. Acad. Sci. USA 77:5201,1980, using a full length mouse SPL cDNA probe labeled by randomlabeling technique (Cobianchi and Wilson, Meth. Enzymol. 152:94–110,1987). This analysis revealed a pattern of expression consistent withthe known SPL activity in various mouse tissues, providing furtherconfirmation that this sequence encodes mouse SPL (FIG. 7).

Example 3 Isolation and Characterization of Human SPL cDNA

This Example illustrates the identification of an endogenous human cDNA.

An EST database was searched using the mouse SPL sequence describedherein. Two distinct EST sequences having strong homology to the mousesequence were identified from human sources. One of these sequencescorresponded to the C-terminus, and the other corresponded to theN-terminus. Primers were designed based on these sequences, and a DNAfragment was amplified by PCR from a human expression library made fromhuman glioblastoma multiforme tissue RNA. The fragment was sequenced andwas shown to contain a deletion, so the primers were used to amplify thegene from human fibroblast RNA. This gene has the sequence provided inSEQ ID NO:3, and the sequence of the gene containing the deletion isprovided in SEQ ID NO:9.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for the purposeof illustration, various modifications may be made without deviatingfrom the spirit and scope of the invention.

1. An isolated antibody specific for a polypeptide having a sequencerecited in any one of SEQ ID NOs: 2, 4 or
 6. 2. A monoclonal antibodyspecific for a polypeptide having a sequence recited in any one of SEQID NOs: 2, 4 or
 6. 3. An antibody according to claim 1 or claim 2,wherein the antibody inhibits the ability of a polypeptide having asequence recited in any one of SEQ ID NOs: 2, 4 or 6 to degradesphingosine-1-phosphate.
 4. A method for detectingsphingosine-1-phosphate lyase in a sample, comprising: (a) contacting asample with an antibody according to claim 1 or claim 2 under conditionsand for a time sufficient to allow the antibody to bind tosphingosine-1-phosphate lyase; and (b) detecting in the sample thepresence of sphingosine-1-phosphate lyase bound to the antibody.
 5. Akit for detecting sphingosine-1-phosphate lyase in a sample, comprisingan antibody according to claim 1 or claim 2 and a buffer or detectionreagent.